We have uncovered a role of the RNA Induced Silencing Complex (RISC) in Drosophila sex determination. RISC components interact strongly with the sex determination master-switch gene, Sex-lethal (Sxl), impacting the female sex determination process. The timing as well as the strength of expression of the specialized X:A ratio sensitive promoter of Sxl, SxlPe, whose activation determines sexual identity, is altered. The proposed studies will: 1) Analyze the basis for the difference in timing and levels of SxlPe activation. 1a) Quantify the RNA of X:A ratio genes as well as SxlPe. We plan to assess the timing of expression of the key known genes which read the X:A ratio in RISC mutant backgrounds, during the period the ratio is being read. The RNA levels of Sxl and these genes will also be analyzed by quantitative RT-PCR. 1b) Determine the role of Heterochromatin protein 1 (HP1) in the activation of Sxl, and how it is linked to RISC. RISC has been shown to influence chromatin compaction and the recruitment of HP1. The distribution of HP1 and other heterochromatin proteins across Sxl and how they affect SxlPe transcription will be examined. 2) Examine the role of the male specific lethals (msl), rox1 RNA and RISC in Sxl activation. We find that dicer2 and maleless act antagonistically with respect to SxlPe expression. Besides maleless, several other msl genes interact with the numerator genes to compromise female viability, and in situs show they fail to correctly activate SxlPe. This effect has a strong maternal component and, surprisingly, includes the rox genes which have no significant full-length RNA in females. We will examine: a) Interactions between dcr2, the male specific lethals with SxlPe In situ analysis will determine whether the other msls also antagonize dicer2 in SxlPe activation. To determine which part(s) of rox are important for Sxl expression, a deletion series of rox1 will be tested as well as an artificially constructed short segment of rox2 which can assemble the dosage compensation complex. b) identify and clone small rox RNAs The maternal effect of the rox genes on Sxl suggests the existence of small rox RNAs. We plan to identify them by Northern blot analysis and propose a method to clone them. 3) determine whether Piwi regulates Rox RNA cleavage Immunoprecipitates of Piwi protein show the presence of rox1 RNA;embryos mutant for piwi show elevated levels of rox1 suggesting Piwi may be the nuclease which cleaves rox RNA in the germline. This hypothesis will be tested. PUBLIC HEALTH RELEVANCE: Understanding the RNA interference system and the processes it regulates, will provide a better understanding of the molecular basis of many different diseases and cancers. Components of RNA interference have been found to affect heart development and function, development of hematopoietic lineages, the immune system and be involved in cancers. Working on the role of the RNA induced silencing complex (RISC) in the sex determination pathway will allow us to use this well established system to understand how RISC regulates and fine tunes this developmental pathway, giving us insights into the various regulatory roles of RISC.